Rejuvenating senescent and centenarian human cells by reprogramming through the pluripotent state.

Laboratory of Plasticity of the Genome and Aging, Institute of Functional Genomics, Montpellier Cedex, France.

Abstract

Direct reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) provides a unique opportunity to derive patient-specific stem cells with potential applications in tissue replacement therapies and without the ethical concerns of human embryonic stem cells (hESCs). However, cellular senescence, which contributes to aging and restricted longevity, has been described as a barrier to the derivation of iPSCs. Here we demonstrate, using an optimized protocol, that cellular senescence is not a limit to reprogramming and that age-related cellular physiology is reversible. Thus, we show that our iPSCs generated from senescent and centenarian cells have reset telomere size, gene expression profiles, oxidative stress, and mitochondrial metabolism, and are indistinguishable from hESCs. Finally, we show that senescent and centenarian-derived pluripotent stem cells are able to redifferentiate into fully rejuvenated cells. These results provide new insights into iPSC technology and pave the way for regenerative medicine for aged patients.

Induction of pluripotency in proliferative and senescent 74-yr-old-derived cells. (A, panel 1) Detection of SAHF by indirect immunofluorescence of H3K9me3 (red) and Hoechst (blue) in proliferative (74P), senescent (74S), and transduced senescent 74-yr-old cells (74S inf) by the six factors. Seven days after transduction, no SAHF were detected. (Panel 2) Eighteen days after transduction, proliferation of infected 74S cells was observed (74S inf). Around day 40, distinct colonies were observed. Representative phase-contrast images are shown. (B) Immunodectection of surface markers TRA-1-60 and SSEA-4 on iPSCs colonies derived from 74S- and 74P-year-old cells (three independent clones). (C) Quantitative RT–PCR of expression levels for endogenous pluripotency factors in the iPSCs from 74P and 74S and their parental fibroblasts. H1 and H9 hESCs and iPSC TH 4 were used as controls. Transcript levels were normalized to GAPDH expression. Error bars indicate standard deviations from duplicate experiments. (D) Bisulfite sequencing analysis of OCT4 and NANOG promoter regions showing demethylation in iPSCs from 74P and 74S, as in H9 hESCs, compared with parental fibroblasts. Each column of circles for a given amplicon represents the methylation status of CpG dinucleotides in one clone for that region. Open circles are unmethylated CpGs and closed circles methylated ones. The left numbers of each column indicate CpG localization relative to the transcriptional start site. (E) In vitro differentiation experiments of iPSCs reveal their potential to generate cell derivatives of all three primary germ cell layers. Immunodetection of SMA, MAP2, and FOXA2 markers specific for endoderm, ectoderm, and mesoderm, respectively. Nuclei are stained with Hoechst (blue). Three independent clones are shown.

Rejuvenated features of fibroblasts redifferentiated from iPSCs generated from senescent and aged fibroblasts with a six-factor-based reprogramming strategy. (A) SA-β-gal staining of proliferative (PD 10) and replicative senescence (PD 60–63) fibroblasts redifferentiated from iPSCs (PD 90). The percentage of positive cells is indicated in the inset. Experiments were performed in triplicate (±SD for standard deviation). (B) Restoration of p21CIP1 and p16INK4A protein expression in iPSC-derived fibroblasts is triggered during replicative senescence. Western blot analysis using β-actin as the loading control. (C) Nonsupervised hierarchical clustering with gene expression profiles of iPSCs redifferentiated into fibroblasts compared with their 74P-, 74S-, and 96-yr-old parental fibroblasts, postnatal fibroblasts, and embryonic fibroblasts differentiated from H1 hESCs.